Fluorescent dyes have many uses and are known to be particularly suitable for biological applications in which the high detectability of fluorescence is required. Examples of their utility include use in immunoassays, labeling nucleotides and oligonucleotides for hybridization studies, binding to polymeric microspheres and staining of cells for use in imaging studies. Dyes are also used for selective destruction of cells such as in the technique of photodynamic therapy
Fluorescence useful for such applications is generally initiated by absorption of light from an external, relatively concentrated light source. The sensitivity of these applications is improved by having dyes that have high absorbance of the exciting light and high fluorescence quantum yield. The applications are furthermore improved by having dyes that resist photobleaching by the exciting light and that have spectral wavelengths in a range that avoids the background from contaminants that may be present in the samples. For many biological applications it is useful to have dyes whose fluorescence is not quenched by water, since most biological measurements are made in aqueous solution.
Certain lasers are particularly useful as a concentrated light source for the excitation of fluorescence. These include the argon laser with principal output at 488 nm and 514 nm; helium-neon lasers that can be selected to have maximum output at either 543 nm, 594 nm, or 633 nm; the krypton laser which has significant output at 568 nm and 647 nm; and light emitting diodes, which are commonly available at this time, with output above 660 nm.
A number of dyes have previously been found to be fluorescent, however many of these dyes have characteristics which interfere with their usefulness. For example, many known fluorescent dyes do not have significant absorbance at the desired excitation wavelengths, or are significantly quenched in aqueous solution or are unstable during the illumination.
Dyes derived from dipyrrometheneboron difluoride have many desirable characteristics. Simple alkyl derivatives of the fluorophore 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene have been described by Treibs & Kreuzer, Difluorboryl-komplexe von di- und tripyrrylmethenen, LIEBIGS ANNALEN CHEM. 718, 203 (1968) and by Worries, Kopek, Lodder, & Lugtenburg, A novel water-soluble fluorescent probe: Synthesis, luminescence and biological properties of the sodium salt of the 4-sulfonato-3,3',5,5'-tetramethyl-2,2'-pyrromethen-1,1'-BF.sub.2 complex, RECL. TRAV. CHIM. PAYS-BAS 104, 288 (1985) as being highly fluorescent with spectral properties that are similar to fluorescein with maximum absorbance at about 490 to 510 nm and maximum emission at about 500 to 530 nm. U.S. Pat. No. 4,774,339 to Haugland et al. (1988) ('339 patent) describes 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (dipyrrometheneboron difluoride) dyes including aryl-substituted derivatives that contain reactive groups suitable for conjugation to biomolecules, that have good photostability, and which have fluorescein-like spectra. Neither the earlier references nor the '339 patent does not disclose the subject heteroaryl dyes or enhanced long wavelength fluorescence properties of their dyes.
As described in the '339 patent, and by Pavlopoulos, et al., Laser action from a tetramethylpyrromethene-BF.sub.2 complex, APP. OPTICS 27, 4998 (1988), the emission of the alkyl derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene fluorescent dyes clearly overlaps that of fluorescein. The overlap allows the alkyl derivatives of dipyrrometheneboron difluoride to be used with the same optical equipment as used with fluorescein-based dyes without modification of the excitation sources or optical filters. As a result of the same spectral characteristics, however, the fluorescence of the known class of alkyl-substituted 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacenes is not readily suitable for detection in combination with fluorescein or for use in applications where excitation by longer wavelength sources such as the helium-neon or krypton lasers or light emitting diodes is required.
Heteroaryl-substituted dipyrrometheneboron difluoride dyes described in this invention have significant absorbance and fluorescence at desired wavelengths, high fluorescence in aqueous solution and good photostability and are thus particularly useful as fluorescent dyes. Furthermore, the subject fluorescent dyes are desirable for use in combination with other fluorescent dyes such as fluorescein or alkyl-substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes in that their fluorescence can be selectively determined by their spectral shift to longer wavelengths, particularly their emission at greater than 540 nm. There are no previously reported examples of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes that contain heteroaryl-substituents or that have been described that have such long wavelength spectral properties.